Selective αvβ3 receptor peptide antagonist for therapeutic and diagnostic applications

ABSTRACT

The present invention is related to new peptide antagonists of α v β 3  receptor, designed on the basis of the crystal structure of integrin α v β 3  in complex with c(RGDf[NMe]V) and the NMR structure of echistatin. These peptides are potent and selective antagonists of the α v β 3  receptor and can be used as novel anticancer drugs and/or new class of diagnostic non-invasive tracers as suitable tools for α v β 3 -targeted therapy and imaging.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. Continuation application of Ser. No.12/089,709 filed on Apr. 9, 2008, which is the national phase ofInternational Application PCT/EP2006/009733 filed on Oct. 9, 2006,which, in turn, claims priority to U.S. Provisional Application60/725,294 filed on Oct. 12, 2005, each of which are herein incorporatedby reference in its entirety.

BACKGROUND OF THE INVENTION

Integrins are members of a family of heterodimeric transmembrane cellsurface receptors that play a crucial role in cell-cell and cell-matrixadhesion processes (Hynes, R. O. Cell 1992, 69, 11-25). These receptorsconsist of an α- and a β-subunit, which non-covalently associate indefined combinations (Eble, J. A. Integrin-Ligand Interaction; Springer:Heidelberg, 1997; pp 1-40). Most of them recognize the Arg-Gly-Asp (RGD)triad found in many extracellular matrix proteins (i.e vitronectin)(Serini, G.; et al. A sticky business. Exp. Cell. Res. 2005, in press)and snake venom disintegrins (Ruoslahti, E.; Pierschbacher, M. Cell1986, 44, 517-518; D'Souza, S. E.; et al. Trends Biochem. Sci. 1991, 16,246-250; Gould, R. J.; et al. Proc. Soc. Exp. Biol. Med. 1990, 195,168-171). Even if different integrins recognize different proteinscontaining the RGD sequence, several studies have demonstrated that theamino acid residues flanking the RGD sequence of high-affinity ligandsmodulate their specificity of interaction with integrin complexes.Despite numerous studies reported in the literature, ligand selectivitytoward different integrin subtypes is still a challenging problem mainlybecause most of the 3D-structures of integrin subtypes remain unknown(Marinelli, L.; et al. J. Med. Chem. 2004, 47, 4166-4177).

An extensively studied member of this receptor class is integrinα_(v)β₃. This integrin is strongly expressed on activated endothelialand melanoma cells, in contrast, it is weakly expressed in quiescentblood vessels and pre-neoplastic melanomas (Hood, J. D.; Cheresh, D. A.Nat. Rev. Cancer 2002, 2, 91-100). Along with α_(v)β₅ integrin, α_(v)β₃is reported to be involved in physiological processes includingangiogenesis and tissue repair as well as pathological conditions suchas tumor induced angiogenesis (Eliceiri, B. P.; Cheresh, D. A. J. Clin.Invest. 1999, 103, 1227-1230; Kumar, C. C. Curr. Drug Targets 2003, 4,123-131), tumor cell migration and invasion (Clezardin, P. Cell. Mol.Life. Sci. 1998, 54, 541-548). Despite the fact that both integrinspromote cell attachment to vitronectin and participate in the sameprocesses, they are reported to be structurally designed to respond todifferent signaling events. Previous studies provided evidence thatbFGF-induced angiogenesis is mediated by α_(v)β₃ whereas VEGF-inducedangiogenesis is mediated by α_(v)β₅ (Friedlander, M.; et al. Science1995, 270, 1500-1502). Melanoma cells expressing α_(v)β₃ migrate invitro and metastasize in vivo without the need for exogenous cytokinestimulation (Filardo, E. J.; et al. J Cell Biol. 1995, 130, 441-450).Conversely, tumor cells expressing α_(v)β₅ integrin require a tyrosinekinase receptor-mediated signaling event for motility on vitronectin andin vivo dissemination (Brooks, P. C.; et al J Clin Invest. 1997, 99,1390-1398). While α_(v)β₅ is widely expressed by many malignant tumorcells, α_(v)β₃ has a relatively limited cellular distribution comparedwith that of α_(v)β₅ (Pasqualini, R.; et al. J. Cell Science 1993, 105,101-111; Walton, H. L.; et al. J. Cell. Biochem. 2000, 78, 674-680.).Therefore, in order to target α_(v)β₃-mediated processes for diagnosticor therapeutic purposes, the development of new compounds that candiscriminate between α_(v)β₃ and α_(v)β₅ is required.

To date, various therapeutic candidates, including antibodies (Gutheil,J. C. et al. Clin. Cancer Res. 2000, 6, 3056-3061), small molecules(Miller, W. H.; et al. Drug Discov. Today 2000, 5, 397-408; Marugan, J.J.; et al. J. Med. Chem. 2005, 48, 926-934), peptidomimetics (Sulyok, G.A.; et al. J. Med. Chem. 2001, 44, 1938-1950; Belvisi, L.; et al. Org.Lett. 2001, 3, 1001-1004), and cyclic peptides (Mitjans, F. et al. Int.J. Cancer 2000, 87, 716-723; Dechantsreiter, M. A.; et al J. Med. Chem.1999, 42, 3033-3040) have been clinically evaluated and shown tosuccessfully modulate α_(v)β₃-mediated processes. So far, thepentapeptide cyclo (-Arg-Gly-Asp-D-Phe-NMeVal-), referred to asc(RGDf[NMe]V) (Eskens, F. A.; et al Eur. J. Cancer 2003, 39, 917-926),is one of the most active α_(v)β₃ antagonists reported in theliterature. Previous studies have demonstrated a higher affinity of thispeptide for integrin α_(v)β₃ as compared to α_(v)β₅ and have reportedinhibition of α_(v)β₃-mediated cell adhesion with IC₅₀ values in themicromolar range when assayed in different tumor cell lines (Goodman, S.L.; et al. J. Med. Chem. 2002, 45, 1045-1051).

The crystal structures of the extracellular segment of integrin α_(v)β₃in its unligated state and in complex with c(RGDf[NMe]V) and the dockingstudies on α_(v)β₃ integrin ligands have shown that the maininteractions are between the positively charged arginine and theα-subunit and between the anionic aspartic acid and the β-subunit(Marinelli, L.; et al. J. Med. Chem. 2003, 46, 4393-404; Xiong, J. P. etal. Crystal structure of the extracellular segment of integrin α_(v)β₃ .Science 2001, 294, 339-345; Xiong, J. P.; et al. Science 2002, 296,151-155), and that selectivity between different subunits is achieved bythe RGD sequence conformation. Previous studies also reported thatechistatin, the smallest (49 residues) of the viper (Echis carinatus)disintegrins, is a potent antagonist of the integrins α_(v)β₃, α₅β₁ andα_(IIb)β₃ (Wierzbicka-Patynowski, I.; et al. J. Biol. Chem. 1999, 274,37809-37814) and that the amino acids adjacent to the RGD motif togetherwith the 41-49 C-terminal residues appear to be critical for theselective recognition of integrins. Mutation and photoaffinitycross-linking experiments, and NMR conformational analysis combined withdocking studies Yahalom, D.; et al. Biochemistry 2002, 41, 8321-8331;Saudek, V.; et al. Biochemistry 1991, 30, 7369-7372), have providedevidence that the C-terminal region of echistatin binds to a site withinthe β₃ subunit of the α_(v)β₃ receptor, which is distinct from the sitesthat bind residues flanking the RGD triad in small peptide ligands.

SUMMARY OF THE INVENTION

This invention concerns peptide or peptidomimetic compounds, containingthe Arg-Gly-Asp sequence, as potent and selective antagonists of theα_(v)β₃ receptor: the compounds of the invention may be used as novelanticancer drugs and/or new class of diagnostic noninvasive tracers assuitable tools for α_(v)β₃-targeted therapy and imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Is a schematic representation of the synthesized peptides.including RGDechi, echiL (SEQ ID NO: 1), echi6-19 (SEQ ID NO: 2) andechi11-19 (SEQ ID NO: 3).

FIG. 2. Shows representative inhibition curves obtained from adhesionassays performed in Kα_(v)β₃ cells. Cells were preincubated withincreasing concentrations of c(RGDfV) (panel A, IC₅₀ 0.68 μM) andRGDechi (panel B, IC₅₀ 0.88 μM) for 1 h at 4° C. and then seeded onvitronectin-coated plates. Cells were allowed to adhere for 1 h at 37°C. and finally counted.

FIG. 3. Shows selectivity of RGDechi for α_(v)β₃. Panel A.Representative inhibition curves obtained from adhesion assays performedin Kα_(v)β₅ cells. Cells were preincubated with increasingconcentrations of c(RGDfV) (closed squares) and RGDechi (open squares)for 1 h at 4° C. and adhesion was determined as described in FIG. 2. Theresults are expressed as the percentage of adherent cells consideringthe untreated control sample as 100%. Panel B Representative inhibitioncurves obtained from adhesion assays performed in Kα_(IIb)β₃ cells.Cells were pre-incubated with anti-α_(v)β₃ blocking LM609 monoclonalantibody and subjected to the adhesion assay on fibrinogen (10 μg/mL).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the invention have the following formula (I):

wherein:

AA1 is an alpha amino acid containing at least three functional groups,selected in the group of Cys, Asp, Glu, Lys, Orn, Pen, Dab or Dap;

AA2 is an alpha amino acid containing at least three functional groups,selected in the group of Cys, Asp, Glu, Lys. Orn, Pen, Dab or Dap;

L is a linker sequence consisting of a number of amino acid residuescomprised between 0 and 2, such as the sequence PG;

(Xaa)n is an amino acid sequence in which n ranges from 1 to 3, whichsequence is substantially homologue to that of sequence 28-30 ofEchistatin: MDD.

(Yaa)m is an amino acid sequence in which m ranges from 2 to 9, whichsequence is substantially homologue to that of C-terminus (41-49) ofEchistatin: RNPHKGPAT (SEQ ID NO: 3).

The cyclic structure of the pentapeptide is obtained via the formationof a peptide bond between the CO of AA1 and the NH of AA2.

AA2 is preferably a D-aminoacid.

Preferred (Xaa)n sequence is MDD whereas preferred (Yaa)m sequence isRNPHKGPAT (SEQ ID NO: 3).

The Xaa group is linked to AA2 via formation of an amide bond betweenthe side chain of AA2 and the N-terminal of Xaa.

The peptides of the invention have either free or acetylated aminogroups at the N-terminus and either free or amidated carboxyl groups atthe C-terminal position; one or two more amino acid residues can beadded onto the C-terminal end.

The invention also relates to compounds of formula (I) which arelabelled, either with the use of a chelating group or directly, withradioactive or paramagnetic metals or radioactive halogens and the saltsthereof with physiologically acceptable organic or inorganic bases orwith anions of physiologically acceptable organic or inorganic acids.

For the compounds of the invention which contain amino acids, the aminoacid residues are denoted by single-letter or three-letter designationsfollowing conventional practices. All of the amino acids used in thepresent invention may be either the D- or L-isomer. The L-isomers arepreferred when not otherwise specified. Commonly encountered amino acidswhich are not gene-encoded may also be used in the present invention.

The term “substantially homologous” means that the amino acid sequenceof a particular compound shows a substantial correspondence to the aminoacid sequence of C-terminal sequence of echistatin, in which at leastthree aminoacids in the sequences can be mutated with any aminoacids,preferably by conservative substitutions. The term “any amino acid” usedabove refers to the L and D isomers of the natural amino acids and“non-protein” amino acids commonly used in peptide chemistry to preparesynthetic analogs of natural peptides, such as alpha amino acidssubstituted and not substituted at the alpha and beta positions of the Land D configurations, and unsaturated alpha and beta amino acids.Examples of “non-proten” amino acids are norleucine, norvaline,alloisoleucine, allothreonine, homoarginine, thioproline,dehydroproline, hydroxyproline, pipecolic acid, azetidine acid,homoserine, cyclohexylglycine, alpha-amino-n-butyric acid,cyclohexylalanine, aminophenylbutyric acid, phenylalanine mono anddi-substituted at the positions ortho, meta and para of the aromaticring, O-alkylated derivatives of serine, threonine and tyrosine,Salkylated cysteine, epsilon-alkylated lysine, delta-alkylatedornithine, aromatic amino acids, substituted at the positions meta orpara of the ring such as phenylalanine-nitrate, -sulfate, -phosphate,-acetate, -carbonate, -methylsulfonate, -methylphosphonate,tyrosine-sulfate, -phosphate, -sulfonate, -phosphonate,para-amido-phenylalanine, C-alpha,alpha-dialkylated, amino acids such asalpha,alpha-dimethylglycine (Aib), alpha-aminocyclopropanecarboxylicacid (Ac3c), alpha-amino cyclobutane-carboxylic acid (Ac4c),alphaminocyclopentanecarboxylic acid (Ac5c),alpha-aminocyclohexanecarboxylic acid (Ac6c), diethylglycine (Deg),dipropylglycine (Dpg), diphenylglycine (Dph). Examples of beta-aminoacids are beta-alanine (beta-Ala), cis and trans 2,3-diaminopropionicacid (Dap). Other non-protein amino acids are identified on the websitehttp://CHEMLIBRARY.BRI.NRC.CA/.

The peptide or peptidomimetic compounds of the present invention can besynthesized by conventional methods used in ordinary peptide chemistry,as described, for example, in M. Bodansky and M. A. Ondetti, PeptideSynthesis, published by Interscience Publishing Co., New York, 1966; F.M. Finn and K. Hofmann, The Proteins, volume 2, edited by H. Neurath, R.L. Hill, Academic Press Inc., New York, 1976; Nobuo Izumiya et al.,Peptide Synthesis, published by Maruzen Co., 1976; Nobuo Izumiya et al.,Fundamental Peptide Synthesis and Experiment, published by Maruzen Co.,1985; Lecture Series on Biochemical Experiment, edited by theAssociation of Biochemistry, Japan, volume 1, “Chemistry of Protein IV”,chapter II, Haruaki Yajima, Peptide Synthesis, 1977.

The peptide can be synthesized by selecting the liquid phase method orthe solid phase method, depending on the structure of the peptide. Thepeptide compounds can also be synthesized by combining the solution andthe solid phase methods.

The compounds of the invention are purified by reverse-phase highpressure liquid chromatography. The compounds are identified by massspectrometry, amino acid analysis, NMR spectroscopy.

The invention also refers to compounds of general formula (II):A-[Y]_(Z)—C  (II)

wherein A is a peptide of general formula (I); z is an integer between 0and 5; Y is a spacer chain respectively bonded to one of thefunctionalities present on the side chains of the individual amino acidspresent in peptide A, or to an N-terminal (—NH₂) group or a C-terminal(—CO₂H) group of A, and to C; when z is an integer between 2 and 5,units Y may be the same or different from each other;

Y is preferably a group of formula (III):

wherein:

r, 1, and q are each independently 0 or 1, and p are an independently aninteger from 0 to 10, provided that at least one of 1, r and q is otherthan zero;

R is hydrogen;

R1 is a hydrogen or a —CH₃ group;

C may be a chelating agent, covalently bound to the spacer Y or directlyto peptide A, or to more than one amino acid units of peptide A, whichis able to complex a paramagnetic metal or a radioisotopes.

Preferred chelating groups are selected from the group consisting of:

a residue of a polyaminopolycarboxylic acid and the derivatives thereof,in particular selected from diethylenetriaminopentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (D03A),[10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid (HPDO3A),4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazamidecan-13-oicacid (BOPTA),N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethylglycine (EOB-DTPA),N,Nbis[2[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]-glycine(DTPA-BMA), 2-methyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetiacid (MCTA),(α,α′,α″,α′″)-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraceticacid (DOTMA); or is the residue of a polyaminophosphate acid ligand orderivatives thereof, in particularN,N′-bis-(pyridoxal-5-phosphate)ethylenediamine-N,N′-diacetic acid(DPDP) and ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP); oris the residue of a polyaminophosphonic acid ligand and derivativesthereof, or polyaminophosphinic acid and derivatives thereof, inparticular 1,4,7,10-tetraazacyclodo decane-1,4,7,10-tetrakis[methylene(methylphosphonic)]acid and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene-(methylphosphinic)acid; or is the residue of macrocyclic chelants such as texaphrines,porphyrins, phthalocyanines; or isN,N-bis[2-[bis(carboxymethyl)amino]ethyl]L-glutamic acid (DTPA-GLU) orDTPA conjugated with Lys (DTPA-Lys). Other chelating groups are reportedin the publication “Radionuclide Peptide Cancer Therapy” edited by M.Chinol and G. Paganelli, Taylor & Francis CRC Press, 2006 (ISBN:0824728874).

C may also be a radiotracer for nuclear medicine, such as F18-galactogroup, covalently bound to spacer Y or directly to peptide A, or to morethan one amino acid unit of peptide A. Other radiotracers are thosereported in the publication “Radionuclide Peptide Cancer Therapy” editedby M. Chinol and G. Paganelli, Taylor & Francis CRC Press, 2006 (ISBN:0824728874).

These peptides can be used in MRI and in Nuclear Medicine applicationfor the diagnosis and treatment of cancers.

The invention accordingly provides also method of treatment of adisorder comprising the administration to an individual suffering fromsuch disorder of a therapeutically effective amount of the α_(v)β₃antagonist of formula I or II.

Examples of specific disorders include pathologies are related toangiogenesis and metastasis, such as breast cancer, musculoskeletaltumors, melanoma, head and neck cancer, human glioma, cervical cancer,vascular restenosis, osteoporosis, rheumatoid arthritis.

More particularly, the compounds of the invention are able to decreasethe proliferation of tumor cells and/or to modulate pathologicangiogenesis.

The compounds of the invention may also be used in the diagnosis ofdisorders using MRI and Nuclear Medicine methods (PET, SPECT etc) byadministering an effective amount thereof to a subject, particularly forthe diagnosis of diseases related to angiogenesis and metastasis such asbreast cancer, musculoskeletal tumors, melanoma, head and neck cancer,human glioma, cervical cancer, vascular restenosis, osteoporosis,rheumatoid arthritis.

The invention also concerns a diagnostic kit comprising the α_(v)β₃antagonist as defined above for the early detection in plasma ofpathologies, such as breast cancer, musculoskeletal tumors, melanoma,head and neck cancer, human glioma, cervical cancer, vascularrestenosis, osteoporosis, rheumatoid arthritis.

The invention is illustrated in more detail by the following example.

EXAMPLE

The peptide having the sequence reported in FIG. 1, and hereinafterreferred to as RGDechi, is a bifunctional chimeric molecule containing acyclic RGD motif and a sequence corresponding to echistatin C-terminaltail connected by a linker. In order to evaluate the activity of thebifunctional molecule RGDechi, echiL (SEQ ID NO: 1), echi11-19 (SEQ IDNO: 3) and echi6-19 (SEQ ID NO: 2) (FIG. 1) were designed. Echi11-19(SEQ ID NO: 3) and echi 6-19 (SEQ ID NO: 2) encompass the 11-19 and 8-19RGDechi sequences respectively, and echiL (SEQ ID NO: 1) corresponds tothe linear precursor of RGDechi.

Synthesis: All peptides were synthesized on a ABI433A automated peptidesynthesizer using Fmoc solid-phase strategy (0.25 mmol). The synthesiswas carried out with Novasyn TGA resin (substitution 0.29 mmol g⁻¹),using all standard amino acids except for Fmoc-D-Glu-OAll in order toinsert the D-Glu⁵ residue in the peptide sequence by its carboxyl sidechain. The first amino acid was bound onto the resin by treatment withFmoc-Thr(tBu)-OH (5 eq)/MSNT (5 eq)/MeIm (3.75 eq) in DCM for 3 h. TheFmoc deprotection step was performed with 30% piperidine in DMF for 10min and active ester coupling reactions were carried out under afourfold excess of amino acid and HBTU (4 eq)/HOBt (4 eq)/DIPEA (8 eq)in DMF (Fields, C. G.; et al Pept. Res. 1991, 4, 95-101). Each couplingwas repeated twice for 1 h followed by a capping step (5 min) performedwith acetic anhydride/DIPEA/DMF (2.6:4.8:92.6 v/v/v).

After the Arg¹¹ coupling reaction an aliquote of the peptidyl-resin wasremoved to yield the echi11-19 peptide. At the end of the Met⁶ coupling,another part of the resin was removed to obtain echi6-19 peptide. Duringthe RGDechi synthesis before the Fmoc deprotection of Lys¹, selectiveα-carboxyl deprotection of D-Glu⁵ residue from allyl group was carriedout by treatment of the peptidyl-resin with PhSiH₃ (24 eq)/Pd(PPh₃)₄(0.25 eq) in DCM. Before the final cyclization the resin was divided intwo parts, in order to obtain echiL and to continue the synthesis ofRGDechi. The cyclization between αNH of Lys¹ and αCO of D-Glu⁵ wasperformed with PyBop (1.5 eq)/HOBt (1.5 eq)/DIPEA (2 eq) in DMF.

The peptides were cleaved off the resin and deprotected using a mixtureof TFA/H₂O/EDT/TIS (94:2.5:2.5:1 v/v/v/v). The resins were then filteredand the peptides were precipitated using cold anhydrous diethyl ether.

The crude products were purified by preparative RP-HPLC on the ShimadzuLC-8A system, equipped with an Uv-V is detector SPD-10 A using aPhenomenex C18 column (21×250 mm; 15 μm; 300 A) and a linear gradient ofH₂0 (0.1% TFA)/CH₃CN (0.1% TFA) from 5% to 70% of CH₃CN (0.1% TFA) in 30min at flow rate of 20 mL/min. The purified peptides were characterizedusing MALDI-TOF spectrometry on a MALDI-TOF Voyager-DE (PerseptiveBiosystem) spectrometer, which gave the expected molecular ion peaks[M-H]+ of 2061.2, 978.1, 1493.6, 2079.2 for RGDechi, echi11-19 (SEQ IDNO: 3), echi 6-19 (SEQ ID NO: 2) and echiL (SEQ ID NO: 1), respectively.

All peptides were synthesized by the solid-phase method using Fmocchemistry. All aminoacids were coupled according to the HBTU/HOBt/DIPEAprocedure (Fields, C. G.; et al Pept. Res. 1991, 4, 95-101). Finaldeprotection and cleavage form the resin were achieved with TFA andscavengers. During the RGDechi synthesis, before the Fmoc deprotectionof Lys¹, α-carboxyl selective deprotection of the D-Glu⁵ residue fromthe allyl group was carried out by treatment of the peptidyl-resin withPhSiH₃/Pd(PPh₃)₄/DCM (Dangles, O. et al. J. Org. Chem. 1987, 52,4984-93). Before the final cyclization, the resin was split in twoparts, to obtain echiL and to continue the synthesis of RGDechi. Thecyclization between the αNH group of Lys¹ and the αCO group of D-Glu⁵was performed with PyBop/HOBt/DIPEA (Coste, J. et al. Tetrahedron Lett.1990, 31, 205-8) in DMF.

The purity and the identity of the peptides were confirmed by analyticalRP-HPLC and MALDI-TOF mass spectrometry. The overall yields of RGDechi,echiL (SEQ ID NO: 1), echi6-19 (SEQ ID NO: 2) and echi11-19 (SEQ ID NO:3), purified by preparative RP-HPLC, were 24%, 30%, 54% and 58%,respectively.

Cell Adhesion and Competitive Assay: Human erythroleukemia K562 cells,stably cotransfected with cDNA of α_(v) or α_(IIb) subunit and β₃ or β₅subunits, were kindly provided by Dr. S. D. Blystone (SUNY UpstateMedical University, Syracuse, N.Y.). Cells were maintained in Iscove'sModified Dubecco's Medium (IMDM) supplemented with 10% heat-inactivatedfetal bovine serum, 100 IU/ml penicillin, 50 μg/mL streptomycin and 500μg/mL G418 in a humidified incubator with 5% CO₂ at 37° C. Expression ofα_(v)β₃, α_(v)β₅ and α_(IIb)β₃ in the transfected clones was confirmedby flow cytometry using FITC-labeled LM609, P1F6 and A2A9/6 monoclonalantibodies. Cells were used to determine affinity for α_(v)β₃ andcross-reactivity with α_(v)β₅ and α_(IIb)β₃ integrin. RGDechi was testedfor its ability to inhibit cell adhesion to immobilized vitronectin orfibrinogen and to compete for the binding with ¹²⁵I-labeled cyclic RGDpeptide. Other cyclic peptides, such as c(RGDfV) and its variantc(RGDyV) as well as specific sequence of RGDechi, were used forcomparison.

Cell adhesion assays were performed as previously described(Chiaradonna, F.; et al EMBO J. 1999, 18, 3013-3023). Briefly, 24-wellflat-bottom plates were incubated overnight with 5 μg/mL vitronectin.The pre-coated plates were rinsed with PBS, incubated for 1 h at 23° C.with 1% heat-denatured bovine serum albumin, and rinsed again. Then,α_(v)β₃ overexpressing K562 cells were incubated with variousconcentrations of cyclic RGD peptides or diluents for 1 h at 4° C.Peptide-treated cells (0.2-0.5×106/100 μL/well) were seeded ontopre-coated plates and allowed to adhere for 1 h at 37° C. in 5% CO₂.Non-adherent cells were removed with gentle washing, whereas adherentcells were detached by trypsinization and counted. Three differentadhesion assays were performed in duplicates. The results of each assaywere analysed by GraphPad Prism Software Inc, San Diego, Calif., usingthe nonlinear regression least-squares method, to estimate the IC₅₀values for each peptide.

To test the selectivity of the novel peptide, α_(v)β₅ or α_(IIb)β₃overexpressing K562 cells were incubated with increasing concentrationof c(RGDfV) or RGDechi and cell adhesion was determined as previouslydescribed using vitronectin or fibrinogen as indicated.

The peptide c(RGDyV) was labeled with ¹²⁵I using the Iodo-Gen method aspreviously described (Del Vecchio, S. et al. Cancer Res. 1993, 53,3198-3206). Briefly, 100 μg of peptide were reacted with 500 μCi of Na¹²⁵I and 12 μg of Iodo-Gen. After 15 min the reaction was stopped by theaddition of 1 μmol of N-acetyltyrosine. The radiolabeled peptide waspurified by unbound iodide by size-exclusion polyacrylamidechromatography (Felding-Habermann, B. Pathophysiol. Haemost. Thromb.2003, 33 Suppl 1, 56-58). The radiolabeled product contained less than3% of free iodide as assessed by HPLC. Cells (1×10⁶) were then incubatedwith ¹²⁵I-labeled c(RGDyV) in the presence or absence of a large excessof unlabeled competitors for 1 h at 4° C. After extensive washing,cell-associated radioactivity was determined by γ-counter.

All synthesized peptides were tested for their ability to inhibit celladhesion to vitronectin. Human erythroleukemia K562 cells overexpressingα_(v)β₃ (Kα_(v)β₃) were incubated with increasing concentrations of thetested peptide and then allowed to adhere to vitronectin-coated plates.Both c(RGDfV) (Sulyok, G. A.; et al. J. Med. Chem. 2001, 44, 1938-1950;Dechantsreiter, M. A.; et al J. Med. Chem. 1999, 42, 3033-3040), ac(RGDf[NMe]V) analogue with comparable biological activity, and RGDechiwere able to inhibit adhesion of Kα_(v)β₃ cells to vitronectin. FIG. 2shows representative inhibition curves obtained by incubating Kα_(v)β₃with c(RGDfV) and RGDechi, respectively. The IC₅₀ value for c(RGDfV)ranged between 0.64 μM and 3.48 μM, whereas the IC₅₀ of RGDechi rangedbetween 0.79 μM and 7.59 μM. RGDechi fragments were tested for theirability to inhibit Kα_(v)β₃ cell adhesion. Incubation with 10 μM ofselected amino acid sequences, such as echi 11-19 (SEQ ID NO: 3),echi6-19 (SEQ ID NO: 2) and echiL (SEQ ID NO: 1), failed to inhibit celladhesion, which remained 97.5%, 99.0%, and 89.5% as compared tountreated control cells.

To test the selectivity of binding of the novel peptide RGDechi, □α_(v)β₅ overexpressing cells (Kα_(v)β₅) were used in the adhesion assay.In FIG. 3A representative inhibition curves are reported. While c(RGDfV)was able to efficiently inhibit adhesion of cells to vitronectin,RGDechi did not show any significant inhibitory effect on Kα_(v)β₅ celladhesion, indicating lack of cross-reactivity with α_(v)β₅. In parallelexperiments, α_(IIb)β₃ overexpressing cells were pre-incubated withLM609 monoclonal antibody and then allowed to adhere to fibrinogen inthe presence or absence of the selected peptide. FIG. 3B shows that,while c(RGDfV) was able to efficiently inhibit adhesion of cells tofibrinogen, RGDechi did not show any significant inhibitory effect onα_(IIb)β₃ overexpressing cells.

Consistent results were obtained from competition binding experimentsindicating that the novel peptide RGDechi efficiently competes with ac(RGDf[NMe]V) analogue labeled with ¹²⁵I [c(RGDyV)] for the binding toα_(v)β₃ overexpressing cells and not to α_(v)β₅ overexpressing clones.

In conclusion, the above results show that the RGDechi chimeric peptideis a novel and selective ligand for α_(v)β₃ integrin.

The invention claimed is:
 1. An antagonist compound of α_(v)β₃ integrin,displaying a selective affinit for α_(v)β₃ integrin, containing a cyclicRGD motif and two echistatin C-terminal moieties covalently linked by aspacer sequence having the following formula (I):

wherein: AA1 is an alpha amino acid selected in the group of Cys, Asp,Glu, Lys, Orn, Pen, Dab or Dap; AA2 is an alpha amino acid selected inthe group of Cys, Asp, Glu, Lys, Orn, Pen, Dab or Dap; L is a linkersequence consisting of a number of amino acid residues comprised between0 and 2, such as the sequence PG; (Xaa)n is an amino acid sequence inwhich n ranges from 1 to 3, which sequence is substantially homologue tothat of sequence 28-30 of Echistatin: MDD (Yaa)m is an amino acidsequence in which m ranges from 2 to 9, which sequence is substantiallyhomologue to that of C-terminus (41-49) of Echistatin: RNPHKGPAT(SEQ IDNO:3).
 2. A pharmaceutical composition comprising a therapeuticallyeffective amount of the α_(v)β₃ antagonist compound of claim 1 and apharmaceutically acceptable excipient.
 3. A pharmaceutical compositioncomprising a therapeutically effective amount of the compound of claim 1and a pharmaceutically acceptable excipient.
 4. The antagonist compoundaccording to claim 1, wherein the cyclic RGD motif is a cyclic peptidein which the ring consists solely of amino-acid residues in peptidelinkages.
 5. The antagonist compound according to claim 1, wherein thecyclic RGD motif is obtainable by formation of a peptide bond betweenterminal residues of a linear peptide.
 6. An antagonist compound ofα_(v)β₃ integrin, displaying a selective affinity for α_(v)β₃ integrin,containing a pentapeptide cyclic RGD motif and two echistatin C-terminalmoieties covalently linked by a spacer sequence.
 7. A method of treatinga disorder comprising administering a therapeutically effective amountof the alpha v beta 3 antagonist compound of claim 1 to a patient inneed thereof.
 8. The method of claim 7 wherein the disorder is apathology related to angiogenesis and metastasis, such as breast cancer,musculoskeletal tumors, melanoma, head and neck cancer, human glioma,cervical cancer, vascular restenosis, osteoporosis, rheumatoidarthritis.
 9. The method of claim 7 wherein treatment is aimed atdecreasing the proliferation of tumor cells.
 10. The method of claim 7wherein treatment is aimed at modulating pathologic angiogenesis.
 11. Amethod of treating a disorder comprising administering a therapeuticallyeffective amount of the compound of claim 1 to a patient in needthereof.